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Initiation reactions in the mRNA-dependent reticulocyte lysate
218
Biochimica et Biophysica .4cta, 698 (1982) 218-221 Elsevier Biomedical Press
BBA Report BBA 90014
INITIATION REACTIONS IN THE m R N A - D E P E N D E N T R E T I C U L O C Y T E LYSATE JOHN E. KAY and C. ROBIN BENZIE
Biochemistry Laboratory, School of Biological Sciences, University of Sussex, Falmer, Brighton BN1 9QG (U.K.) (Received February 10th, 1982)
Key words: Protein synthesis," Translation; Initiation; Initiation complex; (Reticulocyte lysate)
Reticulocyte lysates depleted of mRNA by digestion with micrococcai nuclease still show an unexpectedly high rate of formation of 80 S initiation complexes. Formation of these complexes is sensitive to all inhibitors of the normal protein synthesis initiation process tested. Such lysates contain high concentrations of mRNA fragments which can be utilized for initiation, with which exogenous mRNA must compete. As a consequence of this competition, mRNAs that are weak initiators may be translated poorly by this system even at low exogenous mRNA concentrations.
One of the most successful eukaryotic protein synthesizing systems for the translation of exogenous m R N A is the reticulocyte lysate rendered mRNA-dependent by incubation with micrococcal nuclease in the presence of Ca 2+ [1]. The nuclease can then be inactivated by chelation of Ca 2+ . This treatment degrades almost all the endogenous m R N A to fragments too small to direct the synthesis of acid-precipitable protein [1], but although there is also some fragmentation of ribosomal R N A [2] the system retains the capacity to translate exogenous m R N A at a very high rate, comparable to that of the untreated lysate. In the course of attempts to utilize this system to study the early steps of the eukaryotic initiation process in the absence of mRNA, we have found that the residual m R N A fragments can still be utilized by the system for initiation. The high concentration of these residual m R N A fragments and the efficiency with which they are used implies that exogenous m R N A can only be translated in this system under conditions of strong competition. In accordance with the Lodish model [3,4], those mRNAs which are inherently weak initiators may be preferentially affected. The conditions used for the preparation of 0167-4781/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
mRNA-dependent reticulocyte lysates were precisely as originally described by Pelham and Jackson [1], except that the creatine phosphate concentration was 4 mM, and that all the amino acids were added at a concentration of 50 g M except where otherwise indicated. Incubation conditions for cell-free protein synthesis [1] and determination of incorporation into protein [5] were as previously described except that the incubations contained 1 # C i / m l [14C]leucine and a total leucine concentration of l0 #M. To determine initiation complex formation 50-80 #l samples of mRNAdependent lysate were incubated with 0.4-1.0 pmol [35S]methionyl-tRNAf at 30°C under the same conditions as for protein synthesis. The formation of initiation complexes was then determined after sucrose density-gradient centrifugation of the incubation mixtures as previously described [6]. Messenger RNA-dependent lysates prepared as described had extremely low rates of protein synthesis, but translated added rabbit globin m R N A very efficiently. Addition of globin mRNA increased the rate of incorporation of [14C]leucine into protein at least 100-fold. These results are very similar to those reported by Pelham and Jackson [ 1].
219
Several other eukaryotic cellular mRNA preparations have also been translated by this system in our laboratory with sufficient efficiency to allow characterization of the products by gel electrophoresis and autoradiography. However, in no case has the translation efficiency approached that of globin mRNA, even when the system was supplemented with heterologous tRNA [7]. Despite the very low rate of protein synthesis in mRNA-dependent lysates in the absence of exogenous mRNA, such lysates exhibit a very rapid and extensive transfer of added [35S]methionyl-tRNAf to 80 S initiation complexes (Fig. 1A). Addition of high concentrations of globin mRNA (Fig. 1B) or liver mRNA (Fig. 1C) did not significantly increase the labelling of 80 S complexes. In the experiment shown the lysates were incubated with [35S]methionyl-tRNAf for 2rain, but similar resuits were observed after incubation for periods ranging from 0.5 to 6 min, or when the lysates were preincubated for up to 15 rain before addition of [35S]methionyl-tRNAf. One possibility considered was that the conditions used for nuclease treatment might not have
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Fig. 1. Initiation complex formation by mRNA-dependent lysate incubated with or without exogenous m R N A . Lysate (50 #1) was incubated for 2 min at 30°C with 0.9 pmol [ 35S]methionyl-tRNA f (A) without exogenous mRNA; (B) with 4 # g globin m R N A or (C) with 1.8 #g liver poly(A) + mRNA. The top of each gradient is to the right, and the sedimentation positions of 80 S ribosomes and 40 S ribosomal subunits are indicated by arrows.
80 40 10 A
Jl
, 11
J"B
8 )<
6
E 0_ U
4 2
10
20 10 Fraction
20
Fig. 2. Effect of 7-methyl GTP on initiation complex formation. Lysate (80 /~l) was incubated for 5 min at 30°C in the absence (A) or presence (B) of 1.25 m M 7-methyl GTP. 0.9 pmol [35S]methionyl-tRNAt was added to each incubation for a further 1 min at 35°C.
been sufficiently arduous. However, preincubation of the lysate with 4-times the usual nuclease concentration for twice the usual time did not significantly reduce the incorporation of [35S]methionyltRNAf into 80 S initiation complexes. Similar resuits were also obtained whether the lysates were incubated with or without amino acids. The formation of 80 S initiation complexes in reticulocyte lysates requires the presence of mRNA initiation sites, although the formation of the intermediate 40 S complexes does not [8]. Experiments with known inhibitors of eukaryotic initiation were carried out to confirm that the formation of 80 S complexes observed here was proceeding by the normal pathway. Preliminary experiments established that, as in the intact lysate [8], incubation at 0°C allowed maximum formation of 40 S complexes, but that [ 35S]methionyl-tRNAe was only transferred to 80 S complexes when the incubation temperature was raised. Addition of the mRNA 5'-cap analogue 7-methyl GTP, which partially inhibits the initiation of translation in this system [9], also substantially inhibits 80 S complex formation (Fig. 2). Almost 70% inhibition was seen with 1.25 mM 7methyl GTP and over 50% even when the 7-methyl GTP concentration was reduced to 0.45 mM. Simi-
220 8O 4 0
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Fig. 3. Effect of edeine and sparsomycin on initiation complex formation. Lysate (80/zl) was incubated for 5 min at 30°C with or without inhibitors. 0.4 pmol [ 35S]methionyl-tRNA r was then added and incubation continued for a further 3 rain. (A) control; (B) with 1-10 -5 M edeine; (C) with 1.10 -4 M sparsomycin.
lar results were obtained on addition of 1 • 10 -4 M pyrocatechol violet (data not shown), which also prevents m R N A addition to 40 S initiation complexes in this system [6]. Addition of 1 • 1 0 - S M edeine, which allows 40 S complexes to bind to m R N A but prevents the subsequent addition of 60 S subunits [10], completely abolished the formation of 80 S complexes (Fig. 3B). Substantial inhibition was also achieved by preincubation of the m R N A - d e p e n d e n t lysates for 5 min with 1. 1 0 - a M sparsomycin (Fig. 4C). Sparsomycin is an inhibitor of elongation, and does not affect the initiation process in reticulocytes directly, but preincubation of lysates with the drug does inhibit formation of 80 S initiation complexes indirectly as all m R N A initiation sites become blocked with ribosomes during the preincubation [8]. The results presented in this paper demonstrate that there is very rapid formation of 80 S initiation complexes in m R N A - d e p e n d e n t reticulocyte lysates and strongly support the conclusion that these complexes are formed by the normal initia-
tion pathway, which requires the participation of mRNA. Reticulocyte lysates are known to contain a minor m R N A component resistant to nuclease digestion [1], an observation that we have confirmed. However, this accounts for no more than 1% of the total reticulocyte mRNA, an amount which can in no way account for the 80 S initiation complex formation observed here. Study of the formation of 80 S complexes after addition of known amounts of globin mRNA to sparsomycin-blocked lysates showed that a minimum of 1/~g globin m R N A would have been required to give rise to the number of 80 S complexes formed, an amount equivalent to 25-50% of the globin m R N A initially present. The observation that concentrations of 7-methyl G T P which only partially inhibit protein synthesis substantially reduce 80 S complex formation further confirms that the numbers of 80 S complexes seen could not be a consequence of the very low level of residual initiation on this resistant mRNA species. The precise numbers of 80 S complexes formed is uncertain as the exact amount of endogenous methionyl-tRNA r in the lysates is not known, but the total amount was shown to be sufficient to maintain very extensive labelling of 80 S initiation complexes for several minutes in this very active system. It seems altogether more probable that the m R N A actually used for the formation of 80 S initiation complexes are fragments of degraded globin m R N A too short to give rise to acid-precipitable translation products. Partially degraded m R N A molecules can be utilized for initiation by eukaryotic systems [11] but the minimum size of oligonucleotide necessary for this function in eukaryotes is unknown. It would not be surprising if fragments of significant length survived nuclease treatment, perhaps due to protection by ribosomes. If added mRNA has to compete for initiation with endogenous m R N A fragments, the Lodish model [3,4] would predict that the translation of those m R N A which are weak initiators would be preferentially inhibited. This would particularly be the case if the strong natural initiation sites of fl-globin m R N A had survived. Our observation that several other eukaryotic cellular mRNA pre-
221
parations are translated with lower efficiency than globin m R N A could be accounted for in this way, as also could the report [12] that the mRNA-dependent lysate translates the strong fl-globin m R N A much more efficiently than the weak t~globin mRNA, even at low concentrations of exogenous m R N A when competition would not have been anticipated. The high initiation and elongation rates, low nuclease activity and technical convenience of the mRNA-dependent reticulocyte lysate will ensure its continued usefulness as a eukaryotic translation system. It should, however, be appreciated that if its translation products are used to quantitate the various mRNAs present in a heterogeneous population, the concentrations of inefficiently initiating mRNAs may be underestimated, even if care is taken to use low concentrations of exogenous mRNA. We thank Drs. M.J. Clemens, V.M. Pain and S.A. Austin for valuable discussions, and the Medical Research Council and the Science Research Council for financial support.
References 1 Pelham, H.R.B. and Jackson, R.J. (1976) Eur. J. Biochem. 67, 247-256 2 Kennedy, T.D., Hanley-Bowdoin, L.K. and Lane, B.G. (1981) J. Biol. Chem. 256, 5802-5809 3 Lodish, H.F. (1974) Nature 215, 385-388 4 Bergmann, J.E. and Lodish, H.F. (1979) J. Biol. Chem. 254, 11927-11937 5 Kay, J.E. and Benzie, C.R. (1980) FEBS Lett. 121, 309-312 6 Margulies, L.J. and Kay, J.E. (1976) Biochim. Biophys. Acta 435, 152-158 7 Pelham, H.R.B. (1978) Eur. J. Biochem. 85, 457-462 8 Darnbrough, C., Legon, S., Hunt, T. and Jackson, R.J. (1973) J. Biol. 76, 379-403 9 Chu, L.-Y. and Rhoads, R.E. 0980) Biochemistry 19, 184191 l0 Safer, B., Kemper, W. and Jagus, R. (1978) J. Biol. Chem. 253, 3384-3386 11 Pelham, H.R.B. (1979) FEBS Lett. 100, 195-199 12 Stewart, A.G., Lloyd, M. and Arnstein, H.R.V. (1977) Eur. J. Biochem. 453-459
Biochimica et Biophysica .4cta, 698 (1982) 218-221 Elsevier Biomedical Press
BBA Report BBA 90014
INITIATION REACTIONS IN THE m R N A - D E P E N D E N T R E T I C U L O C Y T E LYSATE JOHN E. KAY and C. ROBIN BENZIE
Biochemistry Laboratory, School of Biological Sciences, University of Sussex, Falmer, Brighton BN1 9QG (U.K.) (Received February 10th, 1982)
Key words: Protein synthesis," Translation; Initiation; Initiation complex; (Reticulocyte lysate)
Reticulocyte lysates depleted of mRNA by digestion with micrococcai nuclease still show an unexpectedly high rate of formation of 80 S initiation complexes. Formation of these complexes is sensitive to all inhibitors of the normal protein synthesis initiation process tested. Such lysates contain high concentrations of mRNA fragments which can be utilized for initiation, with which exogenous mRNA must compete. As a consequence of this competition, mRNAs that are weak initiators may be translated poorly by this system even at low exogenous mRNA concentrations.
One of the most successful eukaryotic protein synthesizing systems for the translation of exogenous m R N A is the reticulocyte lysate rendered mRNA-dependent by incubation with micrococcal nuclease in the presence of Ca 2+ [1]. The nuclease can then be inactivated by chelation of Ca 2+ . This treatment degrades almost all the endogenous m R N A to fragments too small to direct the synthesis of acid-precipitable protein [1], but although there is also some fragmentation of ribosomal R N A [2] the system retains the capacity to translate exogenous m R N A at a very high rate, comparable to that of the untreated lysate. In the course of attempts to utilize this system to study the early steps of the eukaryotic initiation process in the absence of mRNA, we have found that the residual m R N A fragments can still be utilized by the system for initiation. The high concentration of these residual m R N A fragments and the efficiency with which they are used implies that exogenous m R N A can only be translated in this system under conditions of strong competition. In accordance with the Lodish model [3,4], those mRNAs which are inherently weak initiators may be preferentially affected. The conditions used for the preparation of 0167-4781/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
mRNA-dependent reticulocyte lysates were precisely as originally described by Pelham and Jackson [1], except that the creatine phosphate concentration was 4 mM, and that all the amino acids were added at a concentration of 50 g M except where otherwise indicated. Incubation conditions for cell-free protein synthesis [1] and determination of incorporation into protein [5] were as previously described except that the incubations contained 1 # C i / m l [14C]leucine and a total leucine concentration of l0 #M. To determine initiation complex formation 50-80 #l samples of mRNAdependent lysate were incubated with 0.4-1.0 pmol [35S]methionyl-tRNAf at 30°C under the same conditions as for protein synthesis. The formation of initiation complexes was then determined after sucrose density-gradient centrifugation of the incubation mixtures as previously described [6]. Messenger RNA-dependent lysates prepared as described had extremely low rates of protein synthesis, but translated added rabbit globin m R N A very efficiently. Addition of globin mRNA increased the rate of incorporation of [14C]leucine into protein at least 100-fold. These results are very similar to those reported by Pelham and Jackson [ 1].
219
Several other eukaryotic cellular mRNA preparations have also been translated by this system in our laboratory with sufficient efficiency to allow characterization of the products by gel electrophoresis and autoradiography. However, in no case has the translation efficiency approached that of globin mRNA, even when the system was supplemented with heterologous tRNA [7]. Despite the very low rate of protein synthesis in mRNA-dependent lysates in the absence of exogenous mRNA, such lysates exhibit a very rapid and extensive transfer of added [35S]methionyl-tRNAf to 80 S initiation complexes (Fig. 1A). Addition of high concentrations of globin mRNA (Fig. 1B) or liver mRNA (Fig. 1C) did not significantly increase the labelling of 80 S complexes. In the experiment shown the lysates were incubated with [35S]methionyl-tRNAf for 2rain, but similar resuits were observed after incubation for periods ranging from 0.5 to 6 min, or when the lysates were preincubated for up to 15 rain before addition of [35S]methionyl-tRNAf. One possibility considered was that the conditions used for nuclease treatment might not have
A 8040
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Fig. 1. Initiation complex formation by mRNA-dependent lysate incubated with or without exogenous m R N A . Lysate (50 #1) was incubated for 2 min at 30°C with 0.9 pmol [ 35S]methionyl-tRNA f (A) without exogenous mRNA; (B) with 4 # g globin m R N A or (C) with 1.8 #g liver poly(A) + mRNA. The top of each gradient is to the right, and the sedimentation positions of 80 S ribosomes and 40 S ribosomal subunits are indicated by arrows.
80 40 10 A
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, 11
J"B
8 )<
6
E 0_ U
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20 10 Fraction
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Fig. 2. Effect of 7-methyl GTP on initiation complex formation. Lysate (80 /~l) was incubated for 5 min at 30°C in the absence (A) or presence (B) of 1.25 m M 7-methyl GTP. 0.9 pmol [35S]methionyl-tRNAt was added to each incubation for a further 1 min at 35°C.
been sufficiently arduous. However, preincubation of the lysate with 4-times the usual nuclease concentration for twice the usual time did not significantly reduce the incorporation of [35S]methionyltRNAf into 80 S initiation complexes. Similar resuits were also obtained whether the lysates were incubated with or without amino acids. The formation of 80 S initiation complexes in reticulocyte lysates requires the presence of mRNA initiation sites, although the formation of the intermediate 40 S complexes does not [8]. Experiments with known inhibitors of eukaryotic initiation were carried out to confirm that the formation of 80 S complexes observed here was proceeding by the normal pathway. Preliminary experiments established that, as in the intact lysate [8], incubation at 0°C allowed maximum formation of 40 S complexes, but that [ 35S]methionyl-tRNAe was only transferred to 80 S complexes when the incubation temperature was raised. Addition of the mRNA 5'-cap analogue 7-methyl GTP, which partially inhibits the initiation of translation in this system [9], also substantially inhibits 80 S complex formation (Fig. 2). Almost 70% inhibition was seen with 1.25 mM 7methyl GTP and over 50% even when the 7-methyl GTP concentration was reduced to 0.45 mM. Simi-
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Fig. 3. Effect of edeine and sparsomycin on initiation complex formation. Lysate (80/zl) was incubated for 5 min at 30°C with or without inhibitors. 0.4 pmol [ 35S]methionyl-tRNA r was then added and incubation continued for a further 3 rain. (A) control; (B) with 1-10 -5 M edeine; (C) with 1.10 -4 M sparsomycin.
lar results were obtained on addition of 1 • 10 -4 M pyrocatechol violet (data not shown), which also prevents m R N A addition to 40 S initiation complexes in this system [6]. Addition of 1 • 1 0 - S M edeine, which allows 40 S complexes to bind to m R N A but prevents the subsequent addition of 60 S subunits [10], completely abolished the formation of 80 S complexes (Fig. 3B). Substantial inhibition was also achieved by preincubation of the m R N A - d e p e n d e n t lysates for 5 min with 1. 1 0 - a M sparsomycin (Fig. 4C). Sparsomycin is an inhibitor of elongation, and does not affect the initiation process in reticulocytes directly, but preincubation of lysates with the drug does inhibit formation of 80 S initiation complexes indirectly as all m R N A initiation sites become blocked with ribosomes during the preincubation [8]. The results presented in this paper demonstrate that there is very rapid formation of 80 S initiation complexes in m R N A - d e p e n d e n t reticulocyte lysates and strongly support the conclusion that these complexes are formed by the normal initia-
tion pathway, which requires the participation of mRNA. Reticulocyte lysates are known to contain a minor m R N A component resistant to nuclease digestion [1], an observation that we have confirmed. However, this accounts for no more than 1% of the total reticulocyte mRNA, an amount which can in no way account for the 80 S initiation complex formation observed here. Study of the formation of 80 S complexes after addition of known amounts of globin mRNA to sparsomycin-blocked lysates showed that a minimum of 1/~g globin m R N A would have been required to give rise to the number of 80 S complexes formed, an amount equivalent to 25-50% of the globin m R N A initially present. The observation that concentrations of 7-methyl G T P which only partially inhibit protein synthesis substantially reduce 80 S complex formation further confirms that the numbers of 80 S complexes seen could not be a consequence of the very low level of residual initiation on this resistant mRNA species. The precise numbers of 80 S complexes formed is uncertain as the exact amount of endogenous methionyl-tRNA r in the lysates is not known, but the total amount was shown to be sufficient to maintain very extensive labelling of 80 S initiation complexes for several minutes in this very active system. It seems altogether more probable that the m R N A actually used for the formation of 80 S initiation complexes are fragments of degraded globin m R N A too short to give rise to acid-precipitable translation products. Partially degraded m R N A molecules can be utilized for initiation by eukaryotic systems [11] but the minimum size of oligonucleotide necessary for this function in eukaryotes is unknown. It would not be surprising if fragments of significant length survived nuclease treatment, perhaps due to protection by ribosomes. If added mRNA has to compete for initiation with endogenous m R N A fragments, the Lodish model [3,4] would predict that the translation of those m R N A which are weak initiators would be preferentially inhibited. This would particularly be the case if the strong natural initiation sites of fl-globin m R N A had survived. Our observation that several other eukaryotic cellular mRNA pre-
221
parations are translated with lower efficiency than globin m R N A could be accounted for in this way, as also could the report [12] that the mRNA-dependent lysate translates the strong fl-globin m R N A much more efficiently than the weak t~globin mRNA, even at low concentrations of exogenous m R N A when competition would not have been anticipated. The high initiation and elongation rates, low nuclease activity and technical convenience of the mRNA-dependent reticulocyte lysate will ensure its continued usefulness as a eukaryotic translation system. It should, however, be appreciated that if its translation products are used to quantitate the various mRNAs present in a heterogeneous population, the concentrations of inefficiently initiating mRNAs may be underestimated, even if care is taken to use low concentrations of exogenous mRNA. We thank Drs. M.J. Clemens, V.M. Pain and S.A. Austin for valuable discussions, and the Medical Research Council and the Science Research Council for financial support.
References 1 Pelham, H.R.B. and Jackson, R.J. (1976) Eur. J. Biochem. 67, 247-256 2 Kennedy, T.D., Hanley-Bowdoin, L.K. and Lane, B.G. (1981) J. Biol. Chem. 256, 5802-5809 3 Lodish, H.F. (1974) Nature 215, 385-388 4 Bergmann, J.E. and Lodish, H.F. (1979) J. Biol. Chem. 254, 11927-11937 5 Kay, J.E. and Benzie, C.R. (1980) FEBS Lett. 121, 309-312 6 Margulies, L.J. and Kay, J.E. (1976) Biochim. Biophys. Acta 435, 152-158 7 Pelham, H.R.B. (1978) Eur. J. Biochem. 85, 457-462 8 Darnbrough, C., Legon, S., Hunt, T. and Jackson, R.J. (1973) J. Biol. 76, 379-403 9 Chu, L.-Y. and Rhoads, R.E. 0980) Biochemistry 19, 184191 l0 Safer, B., Kemper, W. and Jagus, R. (1978) J. Biol. Chem. 253, 3384-3386 11 Pelham, H.R.B. (1979) FEBS Lett. 100, 195-199 12 Stewart, A.G., Lloyd, M. and Arnstein, H.R.V. (1977) Eur. J. Biochem. 453-459